116 Perspectives in Microbiology 



tion to biological investigations, including nitrogen fixa- 

 tion. Among these were superior methods for cultivation 

 of microorganisms, especially aerobes, resulting from the 

 researches on antibiotics; superior methods for isolation 

 and identification of organic compounds by use of paper 

 and column chromatography; and, in particular, superior 

 methods for tracer studies because of the availability of 

 labeled molecules with appropriate instruments for their 

 estimation. 



Let us look along the research pathway, and with hind- 

 sight put in the record knowledge of which at the time we 

 were only dimly aware. The new instruments were of in- 

 terest not only because they were a powerful and sensitive 

 tool but also because they profoundly affected our thinking 

 about the type of evidence necessary to establish a biolog- 

 ical mechanism and resulted in a much closer scrutiny of 

 the foundations of the logic of our experiments (1). They 

 were even to determine the type of research we would do, 

 for obviously with such bright and shiny tools as these, we 

 would tailor our problems to make use of them. So at 

 Madison we dropped our investigations of the enzyme 

 mechanisms and concentrated on biochemical steps in the 

 pathway. Figure 4, illustrating typical apparatus used for 

 such studies, is included as a reminder of how much the 

 instruments we use today are derived from the physical- 

 chemical laboratory rather than the biological. Many of 

 these powerful new techniques have become so common- 

 place that it is sometimes overlooked that the workers of 

 yesterday were bold enough to challenge nature without 

 them. 



Using these recently discovered jinn of the scientific 

 lamp, we compared the biochemistry of the nitrogen fixa- 

 tion process in different agents and obtained several lines 

 of indirect evidence that NH3 and not NH2OH was the 

 end product of the fixation process (16). The ammonia 

 then apparently entered into the amino acid pool by the 



